This proposal addrsses the structure-function relationship of the vitamin k-dependent blood coagulation proteins, with special attention to gamma carboxyglutamic acid. This program, which involves four separate but related projects, has evolved from previous work developed in the previous period. In Project #1, the structure-function relationship of Factor IX and prothrombin will be studied. Mutant forms of these proteins, obtained from patients with hereditary structural and functional abnormalities of these proteins, will be purified using conformation-specific antibodies, fragmented by proteolysis, and the mutant peptides sequenced. The molecular defect in each mutant protein will be established and correlated to functional defects. In Project #2, the newly discovered KC4 platelet membrane protein will be fully characterized and localized to specific subcellular organelles. The function and binding properties of this protein, expressed only on activated platelets, will be determined. Project #3 will define the role of Gla in prothrombin. A new model of Gla, metal, and membrane interaction in prothrombin using immunochemical and spectroscopic approaches will be evaluated. The membrane binding properties and Gla distribution of a series of partially carboxylated prothrombins should give insight into this problem. Project #4 is a direct clinical application of our immunochemical studies. Using antibodies specific for the fully carboxylated prothrombin (NPT) and des-gamma-carboxyprothrombin (APT), we will continue a clinical trial to evaluate these assays as an improved method of monitoring oral anticoagulant therapy. Vitamin K deficiency in newborns and their mothers will be studied, with the hope of developing simple rational approaches to eliminating hemorrhagic disease of the newborn. Furthermore, assay of the KC4 antigen, specific for activated platelets, will be explored as a method of detection of the prethrombotic state. These basic and clinical projects should make contributions to the understanding of this group of proteins, provide new modalities for the diagnosis of the prethrombotic state and vitamin K dificiency, and develop further data to justify the use of the NPT assay over the prothrombin time significantly reduce the morbidity/mortality associated with oral anticoagulation therapy.